1. Volume 41, Issue 1, Pages 116-126 (July 2014)
Serial Transfer of Single-Cell-Derived Immunocompetence Reveals Stemness of CD8+ Central Memory T Cells 
Patricia Graef, Veit R. Buchholz, Christian Stemberger, Michael Flossdorf, Lynette Henkel, Matthias Schiemann, Ingo Drexler, Thomas Höfer, Stanley R. Riddell, Dirk H. Busch 
Immunity 
Volume 41, Issue 1, Pages (July 2014)
DOI: /j.immuni
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2. Figure 1
Long-Lived Memory after Acute Infection Consists of CD8+CD44hi Central Memory and Effector Memory T Cells
Progenies derived from 500 adoptively transferred naive OT-I CD45.1+ T cells or endogenous H2-Kb/SIINFEKL-Streptamer+ populations detected in spleen of C57BL/6 recipients at day 8 (d8), d28, d100, and d500 postinfection (p.i.) with Lm-OVA (5 × 103 cfu) and analyzed for CD44 and CD62L expression.
(A) Progenies derived from OT-I T cells (pregated on CD45.1+CD8+CD19− cells, upper dot plot) or from polyclonal SIINFEKL-specific endogenous CD8+ T cells (pregated on H2-Kb/SIINFEKL-Streptamer+ CD45.1−CD8+CD19− cells, lower dot plot) recovered at day 500 p.i. and analyzed for CD44 and CD62L expression (two independent experiments, n = 2, gray density plots indicate CD45.1−CD8+ cells).
(B) The CD44hiCD62L+ Tcm and CD44hiCD62L− Tem cell subsets from (A) were further analyzed for CD122 and CXCR3 expression (red histogram: upper panel CD44hiCD62L+ OT-I, lower panel CD44hiCD62L+ endogenous Streptamer+ population; black histogram: upper panel CD44hiCD62L− OT-I, lower panel CD44hiCD62L− endogenous Streptamer+ population; gray histogram: CD44loCD45.1−CD8+ cells). Fraction of marker-positive cells within respective subsets is indicated.
(C) CD44 and CD62L expression of OT-I progenies detected at d8, d28, d100, and d500 p.i. Black dot plots indicate OT-I CD45.1+ T cells (pregated on CD122+CXCR3+ cells), gray density plots indicate CD45.1−CD8+ cells (two independent experiments, n = 2).
Immunity  , DOI: ( /j.immuni )
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3. Figure 2
High Expansion and Differentiation Potential of CD8+ Central Memory T Cells
CD8+ memory T cells were derived from progenies of 500 adoptively transferred naive OT-I CD45.1+ T cells at least 100 days p.i. with Lm-OVA (5 × 103 cfu).
(A) Adoptive retransfer of 100 or 10 CD62L+ (Tcm) or CD62L− (Tem) OT-I CD45.1+ memory T cells analyzed in spleen 8 days p.i. with Lm-OVA (three independent experiments, n100 = 12, n10 = 24). Comparison of recovery rates (unpaired, two-tailed t test, bars indicate mean ± SD), total cell numbers, and total number of Tcm cells generated after transfer of Tcm and Tem cells, respectively (two-tailed Mann Whitney test, median indicated). Recovery rates are defined as fraction of all recipients (n) in which progeny was detectable.
(B) Flow cytometry analysis of peripheral blood from representative recipients in (A) that had received 100 Tcm or Tem OT-I CD45.1+ cells. CD45.1+CD8+ cells were analyzed for expression of CD62L and CD27.
(C) Adoptive retransfer of 10 OT-I CD45.1+ Tcm cells purified from bone marrow (BM), lymph nodes (LNs), or spleen (Spl). Comparison of recovery rates (1-way ANOVA, bars indicate mean ± SD) and relative and absolute sizes of progenies (Kruskal-Wallis test, median indicated) in spleen 8 days after Lm-OVA infection (two independent experiments, n = 16).
Immunity  , DOI: ( /j.immuni )
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4. Figure 3
Similar Developmental Capacity of Single Naive and Central Memory T Cells
Progenies recovered from spleen 8 days after adoptive transfer of single Tn or Tcm OT-I matrix cells and infection with Lm-OVA (5 × 103 cfu).
(A) Absolute number of descendants derived from single Tn (n = 148) and single Tcm (n = 64) cells (six and three independent experiments, respectively, two-tailed Mann-Whitney test, bars indicate median).
(B) Flow cytometry analysis indicates the size as well as the CD27 and CD62L phenotype of three expanded progenies (#1–3) recovered from the same recipient after adoptive transfer of single Tcm OT-I matrix cells.
(C) Scatter plots depict correlation of size and percentage of CD27 or CD62L expression in progenies derived from single Tn (gray) or single Tcm (red) cells (indicated are Spearman correlation coefficients r and respective p values).
(D) Comparison of correlation coefficients (Spearman coefficient and 95% confidence interval) from (C).
Immunity  , DOI: ( /j.immuni )
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5. Figure 4
Serial Adoptive Transfer of Single Cells Demonstrates Self-Renewal Capacity of Individual Central Memory T Cells
(A) Experimental strategy to assess self-renewal capacity of individual CD8+ Tcm cells by successive first-, second-, and third-generation single-cell transfers (SCT) of OT-I CD45.1+ Tn, derived primary Tcm, and derived secondary Tcm cells into C57BL/6 recipients (B6), followed by Lm-OVA infection and blood sampling at d8 postinfection.
(B) Comparison of recovery rates (1-way ANOVA, bars indicate mean ± SD) during successive SCT generations of Tn (first generation) and derived primary Tcm (second generation) and secondary Tcm (third generation) cells (three independent experiments, n1 = 90, n2 = 111, n3 = 100). Recovery rates are defined as fraction of all recipients (n) in which progeny was detectable at peak expansion.
(C) Scatter plots depict correlation in between single-cell-derived progeny size and percentage of CD27- or CD62L-expressing cells at peak expansion. First, second, and third generation SCTs are depicted in gray, red, and blue, respectively (indicated are Spearman correlation coefficients r and significance, n.s. p > 0.05; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001).
(D) Maintenance rates of single-cell-derived progenies (1-way ANOVA, bars indicate mean ± SD). Maintenance rates are defined as the fraction of all single-cell-derived progenies detected at peak expansion, which could also be recovered at ≥2 months p.i.
(E and F) Scatter plots depict percentage (E) and absolute number (F) of Tcm cells in single-cell-derived progenies, harvested from total bone marrow (BM), lymph nodes (LNs), and spleen (Spl) of C57BL/6 recipients, at ≥2 months after first-, second-, or third-generation SCT (Kruskal-Wallis test, median indicated).
Immunity  , DOI: ( /j.immuni )
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6. Figure 5
Degree of Ancestral Expansion Does Not Predetermine Proliferative Behavior of Single Central Memory T Cells
(A) Aligned scatter plots depict the size of progenies derived from individual Tn (large and small gray dots), individual primary Tcm (large and small red dots), or individual secondary Tcm (blue dots) cells, measured in blood 8 days after single-cell transfer (SCT) and Lm-OVA infection. All lines originating from a large dot connect a “mother” population to all of its “daughter” populations. Each daughter population is derived from an individual “maternal” Tcm cell by single cell transfer and infection-driven re-expansion. Serial single cell transfers were always performed at ≥2 months p.i. (three independent experiments).
(B) The correlation plot depicts peak expansion size of mother populations (x axis) versus that of derived daughter populations (y axis) (indicated is spearman correlation coefficient r and respective p value).
(C) Plots depict size of single-cell-derived OT-I CD45.1+ T cells detected in peripheral blood at 8 days p.i. with Lm-OVA. Left: A large and a small “mother” population each derived from an individual Tn cell. Right: Two pairs of large and small daughter populations, each generated by single-cell transfer of single primary Tcm cells derived from large and small mother populations.
Immunity  , DOI: ( /j.immuni )
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7. Figure 6
Single Central Memory T Cells Remain Multipotent throughout Serial Adoptive Transfers
One exemplary single-cell-derived progeny recovered after three successive SCTs of OT-I CD45.1+ T cells. Analysis of the tertiary recipient was performed 12 days after third-generation SCT and infection with Lm-OVA (5 × 103 cfu).
(A) Flow cytometry analysis of single-cell-derived CD45.1+CD8+ progeny size (top) and the respective CD27 and CD62L phenotype (bottom) in bone marrow (BM), liver, lymph nodes (LNs), lung, and spleen (Spl).
(B) Intracellular staining for expression of Eomes and T-bet (black histogram, OT-I; gray histogram, CD44loCD45.1−CD8+ cells). Median fluorescence intensity (MFI) of OT-I T cells (CD45.1+CD8+ cells) is indicated.
(C) Flow cytometry analysis of IFN-γ, IL-2, and TNF-α expression of CD45.1+CD8+ cells after antigen-specific (SIINFEKL peptide) or antigen-independent (PMA/ionomycin) restimulation of tertiary recipient splenocytes.
Immunity  , DOI: ( /j.immuni )
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8. Figure 7
Progeny Derived from Single Central Memory T Cells Can Restore Immunocompetence
(A) Ten Tn or ten tertiary Tcm cells (derived from a single secondary Tcm) were transferred into immunodeficient lymphopenic Rag2−/−Il2rg−/− recipients and prime-boost vaccinated with MVA-OVA (1–2 × 108 pfu i.v.) directly and 12 days after transfer. At 24 days after transfer, mice were challenged with 2 × 105 cfu Lm-OVA i.v. and bacterial burden was determined in spleen 3 days p.i. (three independent experiments, n = 8, two-tailed Mann-Whitney test, median indicated).
(B) Exemplary macroscopic aspect and bacterial culture from spleens of Rag2−/−Il2rg−/− mice from (A) with or without prior transfer of ten Tn or ten tertiary Tcm cells.
(C) Ten tertiary Tcm cells (derived from a single secondary Tcm) were transferred into immunodeficient nonlymphopenic P14tg Rag1−/− mice and prime-boost vaccinated with MVA-OVA (1–2 × 108 pfu i.v.) directly and 12 days after transfer. At 24 days after transfer, mice were challenged with 2 × 106 cfu Lm-OVA i.v. and bacterial burden was determined in spleen 3 days p.i. (two independent experiments, nTcm = 4, nno transfer = 10, two-tailed Mann Whitney test, median indicated).
Immunity  , DOI: ( /j.immuni )
Copyright © 2014 Elsevier Inc. Terms and Conditions